|
| Status |
Public on Feb 28, 2007 |
| Title |
H2O2 perturbation in a glucose-limited chemostat (-0.73 hours) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Cells grown in a glucose-limited chemostat 0.73 hours before the H2O2 perturbation
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
Strain: WT BY4741, haploid
|
| Growth protocol |
Cells were grown in a glucose-limited chemostat (0.02%) and reached steady state. H2O2 (0.6mM) was then inoculated into the chemostat
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were collected, pelleted and immediately frozen.Total RNA was obtained using MasterPure yeast RNA purification kit (Epicenter).
|
| Label |
Cy5
|
| Label protocol |
cDNA was synthesized from total RNA using M-MLV Reverse TranscriptaseRNase H Minus (Promega) and labeled by the indirect amino-allyl method.
|
| |
|
| Channel 2 |
| Source name |
Batch culture cells grown in a glucose-limited medium to late log phase
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
Strain: WT BY4741, haploid
|
| Growth protocol |
Overnight cells (SD) were washed, transferred to Glucose-limited medium (0.02%) and grown to late log phase
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were collected, pelleted and immediately frozen.Total RNA was obtained using MasterPure yeast RNA purification kit (Epicenter).
|
| Label |
Cy3
|
| Label protocol |
cDNA was synthesized from total RNA using M-MLV Reverse TranscriptaseRNase H Minus (Promega) and labeled by the indirect amino-allyl method.
|
| |
|
| |
| Hybridization protocol |
cDNA samples were labeled with Cy3 and Cy5 and combined with blockers: 5?g Herring sperm (Promega), 5?g tRNA (Gibco) and 17.5?g Poly A (Poly A oligos were synthesized with mixed length of 40, 50 and 60 Adenine residues). The labeled cDNAs were concentrated to 40?l using Microcon (Millipore) and 40?l of hybridization x2 solution (10x SSC, 50% formamide and 0.2% SDS) was added. Microarrays containing all yeast ORFs were pre-hybridized by incubating at 42C for 45 minutes in a solution containing 1% BSA, 25% formamide, 5x SSC and 0.1% SDS. The slides were washed in sterile water and dried by centrifugation (3 minutes, 2000 rpm). The labeled samples were boiled for 5 minutes, centrifuged for 1 minute, hybridized on the slide and placed in a hybridization chamber (Corning) for overnight incubation at 42C. The slides were then washed for 5 minutes at 42C with a solution containing 2x SSC and 0.1% SDS. Additional wash was performed at room temperature with a solution containing 0.1x SSC and 0.1% SDS, followed by three additional washes at room temperature in 0.1x SSC solution.
|
| Scan protocol |
Fluorescent array images were collected for both Cy3 and Cy5 using ScanArray 4000 scanner (Packard BioScience)and image intensity data were extracted and analyzed with QuantArray analysis software.
|
| Description |
H2O2 perturbation in a glucose-limited chemostat (-0.73 hours)
|
| Data processing |
Background intensity was subtracted using a Bayesian correction, and the ratios of the two dyes were log2-transformed.Log2 ratios were then corrected for intensity-dependant and spatial-dependant biases by subtracting a Lowess curve followed by a median filter. The two spots corresponding to each gene were then averaged, and genes for which the two spots were significantly different were declared as missing values (along with other genes which were flagged by the image analysis software or removed by manual inspection).
|
| |
|
| Submission date |
Nov 16, 2006 |
| Last update date |
Feb 28, 2007 |
| Contact name |
sagi levy |
| E-mail(s) |
sagi.levy@weizmann.ac.il
|
| Phone |
972-8-9342201
|
| URL |
http://barkai-serv.weizmann.ac.il/GroupPage/
|
| Organization name |
Weizmann Institute of Science
|
| Department |
Molecular Genetics and Physics of Complex Systems
|
| Lab |
Naama Barkai
|
| Street address |
Hertzel
|
| City |
Rehovot |
| ZIP/Postal code |
76100 |
| Country |
Israel |
| |
|
| Platform ID |
GPL4567 |
| Series (1) |
| GSE6302 |
ADH1 deletion cells upon growth on alternative carbon sources; Steady-state grown cells upon environmental perturbations |
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